The influenza virus is responsible for a respiratory disease that may affect up to 20% of the population annually and, on average, kills approximately 20,000 people each year in the United States alone. Symptoms of the virus include sudden fever, chills, headache, myalgia, sore throat and a non-productive cough. Serious respiratory complications, including pneumonia, can develop. Recently, outbreaks of influenza A H5N1 have presented serious disease in humans and birds. Aquatic birds are the natural host for the influenza virus, making it impossible to eradicate the virus. Thus, the need for continued development of influenza treatments and vaccines.
The virus' success in causing disease is due to its ability to evade the immune system by undergoing antigenic change, which is believed to occur when a host is infected simultaneously with both an animal influenza virus and a human influenza virus. Influenza viruses have a segmented genome which contains eight negative-sense RNA (nsRNA) gene segments (PB2, PB1, PA, NP, M, NS, HA and NA) that encode at least 10 polypeptides, including RNA-directed RNA polymerase proteins (PB2, PB1 and PA), nucleoprotein (NP), neuraminidase (NA), hemagglutinin (subunits HA1 and HA2), the matrix proteins (M1 and M2) and the non-structural proteins (NS1 and NS2) (Krug et al., In The Influenza Viruses, R. M. Krug, ed., Plenum Press, N.Y., 1989, pp. 89–152). During reassortment in the host, the human virus may incorporate the animal HA or NA surface protein genes into its genome; thus, producing a new influenza subtype.
In addition to antigenic shift, viruses undergo antigenic drift, via point mutations in the HA and NA surface proteins. This also allows the virus to evade the immune system.
Current Methods for Preparing Vaccines
Until recently, the only influenza vaccines available in the United States were inactivated influenza vaccines. In June 2003, the FDA approved the use of a live attenuated influenza virus vaccine in healthy children and adolescents, 5–17 years of age, and healthy adults, 18–49 years of age. The current vaccines contain two components of influenza A, H1N1 and H3N2, and an influenza B component. Over the last several years, at least one of the components had to be changed each year due to antigenic drift. Clinical isolates of human influenza virus are taken from infected patients and are reasserted in embryonated chicken eggs with a laboratory-adapted master strain of high-growth donor virus, the A/PuertoRico/8/34 (PR8) influenza strain. The purpose of this reassortment is to increase the yield of candidate vaccine strains achieved by recombining at least the HA or NA genes from the primary clinical isolates, with the six internal genes of the master strain donor virus. This provides high growth reassortants having antigenic determinants similar to those of the clinical isolates (Wood, J. M. and Williams, M. S., Textbook of Influenza. Blackwell Science LTd, Oxford, 1998; Robertson et al, Biologicals, 20:213, 1992). Vaccines are prepared by growing this reassorted viral strain in embryonated eggs and then inactivating the purified virus by chemical means.
In the case of a pandemic, chicken eggs will likely be insufficient and suboptimal for influenza vaccine production. Disadvantages of using enbryonated chicken eggs are (1) the lack of reliable year-round supplies of high-quality eggs and the low susceptibility of summer eggs to influenza virus infection (Monto, A S et al., J. Clin Microbiol, 13:233–235, 1981, (2) cultivation of influenza A and B viruses in eggs can lead to the selection of variants characterized by antigenic and structural changes in the HA molecule (Katz, J M, et al., Virology 156:386–395, 1987; Robertson, J S., et al., Virology 143:166–174, 1985; Schild, G C., et al., Nature (London) 303:706–709, 1983), (3) the inability of some viruses to grow in embryonated eggs (Monto AS, et al., J. Clin. Microbiol. 13(1): 233–235, 1981) and (4) the presence of adventitious agents in eggs can jeopardize the preparation of live, attenuated influenza virus vaccines. The use of chicken eggs for inter-pandemic influenza vaccine production requires detailed planning up to six months prior to manufacture to ensure an adequate supply of embryonated eggs (Gerdil, C, Vaccine 21(16): 1776–1779, 2003).
New Methods—Reverse Genetics
Recently developed reverse-genetics systems, based entirely on cDNA, have allowed the manipulation of the influenza viral genome (Palese et., Proc. Natl. Acad. Sci. USA 93:11354, 1996; Neumann and Kawaoka, Adv. Virus Res. 53:265, 1999; Neumann et al., Proc. Natl. Acad. Sci. USA 96:9345, 1999; Fodor et al., J. Virol. 73: 9679, 1999). Furthermore, an eight plasmid system that expresses the nsRNAs from a pol I promoter and the coexpression of the polymerase complex proteins result in the formation of infectious influenza A virus (Hoffmann et al., Proc. Natl. Acad. Sci. USA 97:6108–6113, 2000). This technology allows the rapid production of “custom made” vaccines from cDNA for use in pandemic emergencies. It provides the capability to attenuate pathogenic strains (Subbarao, et al., Virology 305; 192–200, 2003) and eliminates the need to screen reassortant viruses for the 6+2 configuration. The major disadvantage associated with this methodology is the need to use vaccine approved cell lines.
Cell Lines
The commonly used cell lines for rescue of influenza viruses from cDNA are 293T and Madin-Darby canine kidney (MDCK) cells. The 293T cell line is a transformed cell line and is therefore unlikely to be used for human vaccine production, and there are concerns over the tumorogenic potential of MDCK cells (Govorkova E A, et al. J. Virol 70(8): 5519–5524, 2001). Additionally, the utilization of the host specific RNA polymerase I promoters in the reverse genetics systems limits the cell lines to those of human or primate origin.
African green monkey kidney (Vero) cells are characterized, approved and certified by the World Health Organization (WHO) for production of human vaccines. However, Vero cells, while certified, were previously found unsuitable for large-scale production of human influenza virus vaccines. For example, the growth of influenza B in Vero cells was greatly restricted as compared to MDCK cells (Nakamura et al., J. Gen. Virol. 56:199–202,1981). Additionally, attempts to use Vero cells to evaluate the rimantadine sensitivity of human H1N1 and H3N2 influenza A viruses gave ambiguous results, due to the low titers of viruses produced in these cells, as compared with MDCK cells (Valette et al., Antimicrobiol. Agent and Chemotherapy 37:2239–2240, 1993). Thus, these and other studies indicate that influenza viruses have not previously replicated well in Vero cells, making them unsuitable for large-scale vaccine production. (Demidova et al., Vopr. Virosol (Russian) 346–352, 1979); Lau & Scholtissek, Virology 212:225–231, 1995).
The most widely used master strain, A/PuertoRico/8/34, produces a high titer virus in MDCK cells after rescue in MDCK and 293T mixed culture, which is suitable for research purposes. However, it does not produce a high titer virus in Vero cells, mammalian cells approved for use in generating vaccine for humans. By passaging an H1N1 reassortant isolate, A/England/1/53, multiple times in Vero cells, Govorkova EA, et al., J. Infect. Dis. 172(1):250–253, 1995, derived a high-yielding influenza virus. This reassortant virus was reported to contain the surface glycoprotein genes from A/England/1/53 and the remaining genes from A/Puerto Rico/8/34 (H1N1), the commonly used vaccine master strain.
There is a great urgency to develop improved cell culture systems suitable for influenza vaccine production for use in humans, which is a priority for the World Health Organization (Stohr, Vaccine 21:1744–1748, 2003). Therefore, there is the need for a master strain of influenza that will produce high titer influenza virus in cells that have been approved for production of human vaccines.